Bovine myxovirus resistance protein (Mx)1, but not Mx2, mediates antiviral activity against human and avian influenza A viruses, including H5N1 clade 2.3.4.4b
Rubaiyea Farrukee, Yongyan Xia, Rachel Morris, James B. Barnes, Clyde Dapat, Saira Hussain, Yi-Mo Deng, Victoria Meliopoulos, Stacey Schultz-Cherry, Sarah L. Londrigan, Brad P. Gilbertson, Patrick C. ReadingABSTRACT
Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses have recently infected dairy cows in the United States, raising concerns about human spillover. The antiviral roles of bovine host defense factors, including myxovirus resistance proteins (bMx1 and bMx2), remain relatively understudied. We examined bMx1/bMx2 induction in differentiated bovine cells following HPAI H5N1 infection and assessed their antiviral activity using overexpression, knockdown, and mutagenesis approaches. Both bMx1 and bMx2 were more strongly induced in mammary cells than in tracheal cells. Overexpression of bMx1, but not bMx2, significantly inhibited seasonal influenza A viruses (IAVs), low-pathogenic, and highly pathogenic avian influenza viruses, including H5N1 2.3.4.4b, but not influenza B viruses. Conversely, knockdown of bMx1 resulted in enhanced IAV growth. Further, bMx1 inhibited early stages of viral replication, acting to reduce viral transcription and subsequent steps in the viral replication cycle. Known mutations that reduce human MxA (hMxA) activity also impaired bMx1 function, and mutations in the viral nucleoprotein (NP) associated with resistance to hMxA also reduced virus sensitivity to bMx1. Furthermore, phylogenetic analysis showed that a small minority (>0.03%) of avian H5N1 2.3.4.4b viruses already carry some of these NP mutations. These findings suggest that while current H5N1 viruses largely remain susceptible to bMx1, viral evolution or opportunistic reassortment could drive Mx resistance and increase pandemic risk.
IMPORTANCE
Highly pathogenic avian influenza viruses have spilled over into dairy cows and have been circulating on dairy farms in the United States since 2024. The innate antiviral immune responses of cows to influenza viruses have not been extensively studied. In this study, we focused on Mx proteins, a key part of the innate immune defense system toward viruses. We specifically looked at bovine Mx proteins and demonstrated antiviral activity toward different influenza viruses, including the highly pathogenic strains. We also showed that the mechanism of antiviral activity of bovine Mx1 was very similar to that of the human equivalent MxA protein. Further, we found that a small minority of these avian viruses have mutations associated with resistance to both bovine Mx1 and human MxA proteins. Overall, this work highlights the elevated pandemic risk for highly pathogenic influenza viruses through continual circulation in cows, a mammalian species in close proximity to humans.